Vaccinia virus (vv) growth produces plaques two days after infection, providing a rapid in vivo test of viral development. Since inhibition of the activity of the essential vaccinia virus DNA polymerase (vdp) inhibits plaque formation, the effects of alterations of vdp can be tested by plaque assay. In particular, substituted amino acids in the conserved region 6 (r6), which binds deoxyribonucleotides (dNTPs), should inhibit plaque formation, except for substitutions that preserve the structure that allows enzyme function. The goal of this study is to understand the essential local structure that is needed for enzymatic activity by examining the properties of the substituted residues that preserve activity. The effects of allowed substitutions on specific vdp functions can be evaluated by in vitro assays of nucleotide binding and polymerization. To overexpress the enzyme to facilitate the conduct of these assays, the vdp gene was inserted into the DNA of vv vT7lacOI, which contains the T7 RNA polymerase (T7rp) gene. The resultant virus (vdp2) DNA contains 2 copies of the vdp gene, the original and the ectopic copy, whose transcription is enhanced by the T7rp. The T7rp gene and the ectopic gene were activated by isopropyl &#61538;-D- thiogalactopyranoside (IPTG) to initiate overexpression. Also, several vectors were developed to facilitate testing the effect of substitutions in r6, which extends from nucleotides 1987 to 2023 in the transfer plasmid, pv2dp, that contains the vdp gene. A deletion derivative, pv2dpdel, was formed by digesting pv2dp at the unique restriction sites for BstX I (1727) and AflII (2157) to remove a 425-bp dp gene segment (s425). In pv2dpdel, s425 was replaced with a 19-bp linker that restored the restriction sites so that substituted s425 segments could be inserted. Also, pv2dp was used in a polymerase chain reaction (PCR) to synthesize a 469 bp segment , (r6/469), extending from nucleotide 1694 to 2162, that contains sites for Afl II, BstX I, and SnaB I (1974) and BsiHKA I (2027). To use the two last sites, which are not unique in pv2dp, for inserting short segments made from annealed oligonucleotides, the plasmid pACYC177 was modified. pACYC177 has no Afl II, BstX I or SnaB I sites but does have two closely spaced BsiHKA I sites which were enzymatically removed. The resulting larger DNA segment was ligated to form the plasmid pACd. This DNA was cleaved at the unique Hinc II site and ligated to r6/469 to produce the plasmid pAr6/469 which contains unique sites for BstX I, SnaB I, BsiHKA I and Afl II in the dp gene sequence. To obtain a mutant sequence at the dNTP binding site, pAr6/469 was cleaved with SnaB and BsiHKA to release the wt r6 segment and a substituted sequence made from annealed oligonucleotides was inserted. The new plasmid, pAr6/469m1, was digested with BstX I and Afl II and the smaller segment, s425m1, was ligated into pv2dpdel. The resulting plasmid, pv2dpm1 was used to insert the mutated dp gene into vT7lacOI to form the virus vdp2m1 that contains the previously described tyrosine to tryptophan and valine to threonine substitutions at the active site for dNTP binding by the polymerase. When cells infected with vdp2m1 were induced with IPTG before infection and examined for protein production 22 hours post- infection, a prominent band was seen on electrophoretic gels at the dp position. However, as with the wild-type ectopic enzyme, only a portion of the protein was soluble even when incubation was at 31 degrees C. - Vaccinia virus, DNA polymerase, expression, conserved regions